Glycoprotein methods protocols - biotechnology 048-9-273-293.pdf

Glycoprotein methods protocols - biotechnology

The complex O-linked oligosaccharide chains (O-glycans) attached to the

polypeptide backbone of mucins are assembled by glycosyltransferases These enzymes act in the Golgi apparatus in a controlled sequence that is determined by their substrate specificities, their localization in Golgi compartments, and their

relative catalytic activities (1) Activities are controlled by many factors,

includ-ing the membrane environment, metal ions, concentrations of donor and acceptor substrates, cofactors, and, in some cases, posttranslational modifications of enzymes Cloning of glycosyltransferases has revealed the existence of families of homologous glycosyltransferases with similar actions but encoded by different

genes Thus, many steps in the pathways of O-glycosylation appear to be

cata-lyzed by several related glycosyltransferases that may show slight differences in properties and substrate specificities The relative expression levels of these enzymes is cell typespecific and appears to be regulated during the growth and differentiation of cells and, during tissue development, and is altered in many

disease states (2,3).

Figure 1 shows the biosynthetic pathways of O-glycans with the common

mucin O-glycans core structures 1–4 The biosynthesis of other less common core

structures (1) has not been studied in detail Core structures can be elongated

by repeating GlcNAc β1-3Galβ1-4 or GlcNAc1-3Galβ1-3 structures acetyllactosamine chains, i antigens) Poly-N-acetyllactosamine chains may contain

(poly-N-branches of GlcNAc β1-6 residues linked to Gal- (I antigen), and may be terminated

by blood group or tissue antigens (blood group ABO and Lewis antigens) or by sialic acid and sulfate Many of the enzymes involved in these elongation and

termination reactions also act on N-linked oligosaccharides of glycoproteins and

on glycolipids.

From: Methods in Molecular Biology, Vol 125: Glycoprotein Methods and Protocols: The Mucins

Edited by: A Corfield © Humana Press Inc., Totowa, NJ

Fig 1 Pathways of O-glycan biosynthesis The first step of O-glycosylation is

cata-lyzed by polypeptide α-GalNAc-transferase (path a), which acts preferably on Thr in

vitro The occurrence of various O-glycan structures is cell type specific and varies with

cellular activation and differentiation, and in disease states Core 1 and 2 structures arethe most common core structures in mucins, and are synthesized by core 1 β3-Gal-trans-ferase (path b) and core 2 β6-GlcNAc-transferase (path d) Core 3 is synthesized by core 3β3-GlcNAc-transferase (path c) and core 4 by core 4 β6-GlcNAc-transferase (path e).GalNAc- may be sialylated by α6-sialyltransferases (path j) to form sialylα2-6GalNAc-,which cannot be converted to any of the core structures After the synthesis of cores,chains may be elongated, sulfated, fucosylated, or sialylated, and blood group and otherantigenic determinants may be added Core 1 is sialylated by α3-sialyltransferase (path l),and this reaction blocks core 1 branching and elongation with the exception of α6-sialylation by α6-sialyltransferase (path m), which may differ from those catalyzingpath j The α3-sialyltransferase can also act on the Gal residue of core 2 The Gal residue

of core 1 may be sulfated by Gal 3-sulfotransferase (path k) Sulfation will also blockcore 1 elongation and branching Cores 1 and 2 are elongated by elongation β3-GlcNAc-transferase (path h) On galactosylation of the GlcNAc residue of core 2 by β4-Gal-trans-ferase (path f), the poly-N-acetyllactosamine chains can be assembled by the repeatedactions of β4-Gal-transferase and i β3-GlcNAc-transferase (paths f and g, respectively).GlcNAcβ1-6 Gal branches (I antigen) may be introduced into these chains by I β6-GlcNAc-transferase (path i)

Golgi glycosyltransferases have a domain structure characteristic of type II membrane proteins; the amino terminus extends into the cytoplasm, followed by a membrane anchor domain, and a catalytic domain at the carboxy terminus, which extends into the lumen of the Golgi Mainly the membrane anchor and adjacent amino acid sequences, but also other protein determinants of these enzymes, as well as the membrane structure, determine the localization of transferases in vari-

ous Golgi compartments (4) The donor substrates for mucin glycosyltransferases

are nucleotide sugars:

nucleotide-sugar + acceptor → product + nucleotide (1)Mucin sulfotransferases transfer sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the hydroxyls of sugars:

PAPS + acceptor → product + PAP (2)These donor substrates are synthesized in the cytosol, with the exception of CMP-sialic acid, which is made in the nuclear compartment, and are transported into

the Golgi by specific transporter systems (5).

In the sequences of glycosylation reactions, glycosyltransferases often compete for a common substrate For example, the enzymes that synthesize cores 1 and 3

(Fig 1, paths b and c) compete for GalNAc-R substrates Conversely, certain products

formed may block further reactions; for example, no glycosyl transferase acts on sialyl α2-6GalNAc (Fig 1, path j), which therefore blocks extension of chains Alter-

natively, certain reactions may be required prior to further conversions For example, core 1 has to be formed before a GlcNAc β1-6 residue can be added to GalNAc in the

synthesis of core 2 (Fig 1, path d) The distinct specificities of glycosyltransferases

therefore regulate the pathways, and thus the relative amounts of final O-glycan

struc-tures found in mucins The peptide backbones as well as existing glycosylation of

substrates near the O-glycosylation sites also have an important function in regulating

O-glycosylation (6) Thus, primary O-glycosylation as well as the synthesis of various

O-glycan core structures appear to be sitedirected by peptide sequences and their

glycosylation patterns.

Based on many different studies, O-glycosylation appears to be initiated mainly

in early Golgi compartments The first enzyme in the O-glycosylation pathways,

polypeptide α-GalNAc-transferase (Fig 1, path a), has been localized to the cis Golgi compartment in porcine submaxillary gland (7) but can be more broadly dis- tributed throughout the Golgi in other cell types (8) The various members of this

glycosyltransferase family have slightly different specificities toward their peptide substrates, have different cell type–specific expression patterns, and may be local-

ized to different subcellular compartments (9,10) Polypeptide ferase does not require a specific peptide sequence in the substrate; however,

α-GalNAc-trans-particular charged amino acids (11) as well as existing glycosylation (12) influence

the activity.

Most mucins and other glycoproteins contain O-glycans with the core 1 structure,

and the enzyme synthesizing core 1, core 1 β3-Gal-transferase (Fig 1, path b), is a

ubiquitous enzyme (1,13) The activity is a prerequisite for the synthesis of T-antigens and sialylated core 1 structures, as well as core 2 structures (14) The peptide sequences

of glycopeptide substrates and the existing glycosylation determine the activity of core 1

β3-Gal-transferase (15) Erythrocytes from patients with permanent mixed-field polyagglutinability (16), human T-lymphoblastoid Jurkat cells (17), and human colon cancer cells LSC (18) lack the enzyme and therefore cannot make cores 1 and 2 struc-

tures A similar effect can be introduced by the use of GalNAc α-benzyl, which is an

alternative substrate for enzymes extending O-glycan chains, and which can penetrate

cell membranes to compete with endogenous substrates of mucin Thus, cells treated

with this O-glycosylation inhibitor exhibit truncated O-glycans, terminating mainly in

GalNAc (19) These truncated chains no longer carry ligands for cell-cell interactions,

and the binding of colon cancer cells to the endothelium via E-selectin is significantly

reduced (20).

The synthesis of core 2 (Fig 1, path d) is catalyzed by core 2

β6-GlcNAc-trans-ferase (21) Several apparently related β6-GlcNAc-transferases exist that synthesize GlcNAc β1-6 branches on Gal or GalNAc (1,22,23) The L-type core 2 β6-GlcNAc-

transferase occurs in leukocytes and other cells and only synthesizes core 2 The M-type enzyme is found in most mucin-secreting cell types and can synthesize the GlcNAc β1-6 branch of core 2, core 4 (Fig 1, path e), and the I antigen (Fig 1,

path i) (24) The L-enzyme activity increases during cellular activation and entiation (25,26) The M enzyme may be affected in cancer cells (3,27) Core 2

differ-β6-GlcNAc-transferase appears to be localized to cis and medial Golgi

com-partments (27a,28), which is in agreement with its role in synthesizing a central

O-glycan core structure.

The enzymes synthesizing O-glycan cores 3 and 4 (Fig 1, paths c and e,

respec-tively) appear to occur exclusively in mucin-secreting tissues since these cores

have not been found in nonmucin molecules (1) Core 3 is synthesized by core 3 β3-GlcNAc-transferase (29) The enzyme is enriched in colonic tissues but reduced in colon cancer tissue (30,31) and is lacking in many other tissues The activity appar- ently is lacking in colon cancer cell lines (27) The enzyme activity synthesizing

core 4, core 4 transferase, resides in the M-type core 2

β6-GlcNAc-transferase (24,29).

Poly-N-acetyllactosamine chains of mucins are assembled by the repeating actions

of β4-Gal-transferase (Fig 1, path f) (32) and i β3-GlcNAc-transferase (Fig 1, path g) (33) These enzymes are ubiquitous, and may be considered as housekeeping enzymes.

However, their expression is often up- or downregulated in healthy tissues as well as

in a number of disease states (2,3,34) The reaction catalyzed by β4-Gal-transferase

occurs mainly in the trans-Golgi (35) Yet another elongation ferase elongates core 1 and 2 structures, also by a GlcNAc β1-3Gal linkage Fig 1,

β3-GlcNAc-trans-path h) (36).

Poly-N-acetyllactosamine chains may acquire GlcNAc β1-6 (GlcNAcβ1-3) Gal branches in a developmentally regulated fashion, which leads to a change from the i

to the I antigenicity Some of the I β6-GlcNAc-transferases (Fig 1, path i)

synthe-sizing the I branch act on terminal Gal residues whereas others recognize internal

Gal residues (1,37,38) Most of the enzymes synthesizing blood group ABO, Lewis,

and other antigenic determinants act on O- and N-glycans as well as glycolipids By

contrast, sialyltransferases often prefer one type of glycoconjugate (39) Two

sialyltransferase families, α3- and α6-sialyltransferase, act preferably on

mucin-type O-glycans.

α3-Sialyltransferase acts on Gal residues of cores 1 and 2 (Fig 1, path l) (24,40,41) The enzyme is developmentally regulated in thymocytes (42) and increased in leuke- mia cells (43) and several cancer models (3,30) The α3-sialyltransferase has been

localized to medial and trans-Golgi compartments (44) The sialylation reaction

cata-lyzed by this enzyme has an important role in keeping O-glycan chains short and sialylated Since the enzyme acts relatively early in the O-glycan extension pathways

(Fig 1, path l), it has the ability to compete with branching and elongation reactions.

Once core 1 is α3-sialylated, it is no longer a substrate for extension reactions although it can still be converted to the disialylated core 1 by α6-sialyltransferase

(Fig 1, path m).

The α6-sialyltransferase (Fig 1, path j) that acts on GalNAc-R to form the

sialyl-Tn antigen, sialyl α2-6GalNAc-Th/Ser (45), requires glycoproteins as substrate and cannot act on GalNAc-benzyl or nitrophenyl substrates (46,47) However,

another type of α6-sialyltransferase (α6-sialyltransferase III) does not have a tide requirement, but is specific for the α3-sialylated core 1 structure (48) The

pep-disialylated core structure can probably be synthesized by α6-sialyltransferase III and other α6-sialyltransferases (Fig 1, path m) Modifications of the sialic acid

residues of mucins include O-acetylation, catalyzed by specific O-acetyltransferases

acting in the Golgi (49).

The common sulfate ester linkages in mucins are SO4-6-GlcNAc and SO4-3-Gal Several types of sulfotransferases have been described that act on the 6-position of

GlcNAc (50) or the 3-position of Gal of core 1 (Fig 1, path k) and N-acetyllactosamine structures (51,52) Sulfated oligosaccharides appear to play an important role in cell adhesion through binding to selectins and in the control of bacterial binding (53,54).

Sulfation also functions in directing the biosynthetic pathways of complex O-glycans

by blocking certain reactions For example, sulfation of core 1 prevents the branching

reaction to form core 2 (51).

The enzymes catalyzing the reactions depicted in Fig 1 assemble mucin-type

O-linked carbohydrate chains and are listed in Table 1, together with their substrates,

enzyme products, and the high performance liquid chromatography (HPLC) tions of product separation Probably none of these enzymes are specific for mucins,

condi-but also act on other glycoproteins that carry O-glycans, and can act on various peptides with O-linkages In vitro, many of these enzymes utilize synthetic compounds

glyco-as substrates in which the peptide chain is replaced by a hydrophobic group The substrate should be clean, specific, and easy to isolate in order to determine the enzyme activity and specificity accurately For a few enzymes, purified mucins with defined glycosylation are available as substrates However, mucins are usually too heterogeneous in their carbohydrate structures, and therefore the use of synthetic com- pounds with defined structure is preferred In addition, it is much easier to determine

the product structure of synthetic substrates as a proof of the assayed activity When a compound is a potential substrate for several glycosyltransferases present in the enzyme preparation, or several reactions occur in sequence, the various products have to be separated and identified This can usually be achieved by HPLC With the exception of β1,6-GlcNAc-transferases, UDP-sugar binding enzymes require the pres- ence of divalent metal ion for optimal activity Thus, measuring a β6-GlcNAc-trans- ferase activity in the presence of EDTA will eliminate the activity of other GlcNAc-transferases potentially acting on the same substrate Enzymes utilizing CMP-sialic acid may be stimulated by metal ions but usually can act in their absence Unless they are released and secreted, or are produced as soluble recombinant enzymes, glycosyltransferases are membrane-bound enzymes, and their activities are stimulated by detergents.

A convenient way of identifying and quantifying glycosyltransferase products is by the use of nucleotide-sugar donors that contain 14C or 3H-labeled radioactive sugar Similarly, the sulfate moiety of PAPS can be labeled with 35S Calculations of sulfotransferase activities must take into account the relatively short half-life of

6 Small pieces of tissue, or cells

2.2 Preparation of Substrates and Standard Compounds

1 Commercially available oligosaccharides: GlcNAc, GalNAcα-benzyl, Galβ1-3GalNAcα-benzyl, GlcNAcβ1-3 GalNAcα-p-nitrophenyl [pnp], Galβ1-4 GlcNAc,GlcNAcβ1-3 Galβ-methyl (Sigma, St Louis MO); Galβ1-3 GalNAcα-pnp (TorontoResearch Chemicals, Toronto, Canada)

2 Thr-peptides, synthesized by Hans Paulsen, University of Hamburg, Germany

(15,55).

3 Frozen sheep submaxillary glands (Pel-Freez, Rogers, AR) to isolate ovine submaxillary

mucin (OSM), 0.1 N H2SO4, bovine testicular β-galactosidase (Boehringer, Laval,

Canada), 0.1 M Na-citrate buffer, Sephadex G25 column.

4 Components of enzyme assays to prepare product standards enzymatically GlcNAcβ1-3GalNAcα-benzyl, GlcNAcβ1-6 (GlcNAcβ1-3) GalNAcα-pnp, GlcNAcβ1-3 Galβ1-4GlcNAc, GlcNAcβ1-6 (GlcNAcβ1-3 Galβ1-3) GalNAcα-benzyl, GlcNAcβ1-6(GlcNAcβ1-3) Galβ-methyl, sialylα2-3 Galβ1-3 GalNAcα-pnp, SO4-3 Galβ1-3GalNAcα-benzyl, SO4-3 Galβ1-4 GlcNAc

5 Enzymatically prepared substrates: GlcNAcβ1-6 (Galβ1-3) GalNAcα-benzyl, sialylα2-3Galβ1-3 GalNAcα-pnp

6 Nuclear magnetic resonance (NMR) and mass spectrometers, reagents for methylationanalysis.

2.3 Separation and Identification

of Glycosyltransferase and Sulfotransferase Products

1 HPLC apparatus

2 HPLC columns C18, NH2 (amine), PAC (cyano-amine)

3 Acetonitrile/water mixtures

4 Dionex system for high-performance anion-exchange chromatography (HPAEC)

5 Bio-Gel P4 or P2 column (80 × 1.6 cm) (Bio-Rad, Hercules, CA)

6 Ion-exchange columns (AG1 × 8, 100–200 mesh, Bio-Rad)

7 High-voltage electrophoresis apparatus, 1% Na-tetraborate, Whatman No 1 paper

8 C18 Sep-Pak columns, methanol

9 0.05 M KOH/1 M NaBH4 for β-elimination

10 Scintillation fluid, scintillation counter

2.4 Polypeptide α -GalNAc-Transferase Assays

1 5% Triton X-100

2 0.5 M N-morpholino ethanesufonate (MES) buffer, pH 7.

3 0.05 M Adenosine 5'-monophosphate (AMP) to inhibit pyrophosphatases.

4 0.5 M MnCl2

5 10 mM UDP-GalNAc (2000 dpm/nmol) donor substrate.

6 5 mM Acceptor substrate solution: Thr-containing peptide.

7 Enzyme homogenate or solution

2.5. β 3- and β 6-GlcNAc-Transferase Assays

1 5% Triton X-100

2 0.5 M MnCl2 (for β3-GlcNAc-transferases only)

3 0.5 M MES buffer, pH 7.

4 0.05 M AMP.

5 0.5 M GlcNAc to inhibit N-acetylglucosaminidases.

6 50 mM γ-galactonolactone (if substrate with terminal Gal is used) to inhibitgalactosidases

7 10 mM UDP-GlcNAc (2000 dpm/nmol).

8 5 mM Acceptor substrate solution: GalNAcα-benzyl, Galβ1-3 GalNAcα-benzyl, Galβ1-4GlcNAc, GlcNAcβ1-3Galβ-methyl, or GlcNAcβ1-6 (Galβ1-3) GalNAcα-benzyl

9 Enzyme homogenate or solution

2.6 Core 1 β 3-Gal- and β 4-Gal-Transferase Assays

7 5 mM Acceptor substrate solution: GalNAcα-benzyl or GlcNAc.

8 Enzyme homogenate or solution

2.7. α 3- and α 6-Sialyltransferase Assays

1 5% Triton X-100

2 0.5 M Tri-HCl buffer, pH 7.

3 0.05 M AMP.

4 10 mM CMP-sialic acid (2000 dpm/nmol).

5 5 mM Acceptor substrate solution: DS-OSM with 3 mM GalNAc concentration, Galβ1-3GalNAcα-pnp, or sialylα2-3 Galβ1-3 GalNAcα-pnp

6 Enzyme homogenate or solution

7 High-voltage electrophoresis apparatus

5 0.05 M adenosine triphosphate (ATP).

6 0.1 M 2,3-Mercaptopropanol to inhibit PAPS degradation.

glycosidases that degrade substrates and products, and proteases that degrade the peptide moiety of substrates and products may be present, and deactivate the enzyme to be assayed These unwanted reactions can be suppressed with specific inhibitors.

1 To prepare crude homogenate, hand homogenize tissue in 10 times the volume of 0.25 M

sucrose For most studies of crude enzymes, this preparation is sufficient The nate can be stored at –20°C for a few months, or at –70°C for several years If sufficientmaterial is available, a more enriched enzyme fraction can be prepared as microsomes.Microsomes may be prepared from homogenates by first removing a low-speed pellet by

homoge-centrifugation at 10,000g, followed by the precipitation of microsomes from the tant at 100,000g The microsomal pellet is hand homogenized in 10 times the volume of 0.25 M sucrose.

superna-2 Enzymes from cultured cells are prepared similarly Harvest cells from the culture plate,and wash three times with 0.9% NaCl by gently stirring and centrifuging cells After

washing, hand homogenize cells in 0.25 M sucrose (1 mL/108cells) and store as

described in step 1.

3.2 Preparation of Substrates and Standard Compounds

Substrates may be purchased, prepared by chemical synthesis or combined cal-enzymatic synthesis, or prepared by enzymatic synthesis or degradation from natural glycoproteins.

chemi-1 GalNAc-OSM is prepared from purified sheep submaxillary mucin, ovine

submaxil-lary mucin (OSM) (29) Treat OSM with 0.1 N H2SO4for 1 h at 80°C to remove sialicacid and fucose For high purity, follow by digestion with bovine testicular β-galac-

tosidase (56), which removes the small amount of β1-3–linked Gal residues present

in OSM (30).

2 Substrate and product compounds that are not commercially available are synthesizedwith a known source of the desired enzyme under the conditions described for the stan-dard transferase assay

3 Low molecular weight compounds are isolated by gel filtration on Bio-Gel P4 or P2

col-umns, followed by HPLC (Table 1) The purity and linkages of all compounds should be

verified by mass spectrometry (MS) and 1H-NMR The concentrations of individual ars can be determined after acid hydrolysis (1 h at 80°C with 33% trifluoroacetic acid forsialic acid–containing compounds, 1 h at 100°C with 6 N HCl for neutral sugars) by

sug-HPAEC (Dionex system) as described in Subheading 3.4.5.

3.3 Separation and Identification

of Glycosyltransferase and Sulfotransferase Products

To demonstrate that an enzyme activity is synthesizing a certain sugar linkage, the product has to be isolated and its structure determined This is especially important when a new enzyme activity is to be assayed or when a novel variant of a known activity is expected.

1 Produce large amounts of glycosyltransferase product in a standard assay, possibly afterincubation for 8–24 h, and pass through an AG1 × 8 column to remove nucleotide sugar

and negatively charged molecules For sulfotransferase products, run high-voltage trophoresis after the standard assay.

elec-2 Purify low molecular weight compounds by HPLC or using the Dionex system, as

de-scribed in Subheading 3.4.5 Low molecular weight compounds separated by

high-volt-age electrophoresis, can be eluted off the paper with water, by placing the paper intosyringes and centrifuging at low speed Borate is removed by repeated flash evaporationwith methanol

3 Purify mucin substrates by passing incubation mixtures through AG1 × 8 columns,

followed by gel filtration on Bio-Gel P4 or P2 O-glycans are released from mucins

byβ-elimination (0.05 N KOH/1 M Na BH4at 45°C for 16 h) After neutralization,

purify reduced O-glycan-alditols by gel filtration on Bio-Gel P4 or P2 columns, and

by HPLC

4 Carry out structural analysis of all low molecular compounds and alditols by NMR, MS (fast atom bombardment, electrospray, or matrix-assisted laserdesorption ionization), and methylation analysis If small amounts of product areavailable, chromatographic methods, including HPLC and the Dionex system, withthe use of standard compounds, and sequential glycosidase digestion are useful

1 After the incubation, stop the reaction with 100 mL of ice-cold water Apply mixture

to a column (a Pasteur pipet) of 0.4 mL of AG1 × 8, which removes excess tive nucleotide sugar Wash the column three times with 0.6 mL of water and collectthe eluate

radioac-2 Add 5 mL of scintillation fluid and estimate radioactivity with a scintillation counter.Since the radioactivity in the eluate includes free radioactive sugar (originating fromnucleotide sugar breakdown) and radioactive products from various endogenous sub-strates, the radioactivity of assays lacking exogenous substrates has to be substractedfrom the disintegrations per minute obtained The specific enzyme activity is calculated

hydro-to Sep-Pak C18 columns.

1 After the incubation, apply the mixture onto a Sep-Pak C18 column, previously washed

in water Wash columns with 5 mL of water to elute excess nucleotide sugar and freeradioactive sugar